Ng Spot Research Articles

Simultaneous Quantification of Piperine, Vasicine and Eugenol in <i>Kabasura Kudineer</i> by HPTLC

Kabasura Kudineer is a polyherbal decoction of the Siddha medical system (an Indian system of medicine), traditionally used to cure fever, colds, coughs, and respiratory ailments. The government of India had recommended Kabasura Kudineer as one of many preventive/treatment measures for COVID-19. Kabasura Kudineer Choornam is an admixed coarse powder of 15 herbs and its decoction is Kabasura Kudineer. The chemical constituents in the 15 herbs used for the preparation of the Choornam are known but the constituents present in the Kabasura Kudineer (decoction) are unidentified. Piperine, vasicine and eugenol are known for their potent activity against respiratory tract infections; hence, they were selected as marker compounds. The present work was planned to simultaneously quantify piperine, vasicine and eugenol in Kabasura Kudineer by the HPTLC method. The optimised mobile phase was toluene: ethyl acetate: methanol: ammonia (5:9:3:0.5, v/v/v/v), and the scanning was carried out at 287 nm. The Rf values of piperine, vasicine and eugenol were found to be 0.70, 0.32 and 0.82, respectively. The linearity range of piperine and vasicine was 500-3000 ng spot–1 and it was 10–60 ng spot–1 for eugenol. The quantities of piperine, vasicine and eugenol in Kabasura Kudineer (100 mL) were 0.03, 0.056 and 0.035 % w/v, respectively. This developed method can be used to simultaneously quantify piperine, vasicine and eugenol in any polyherbal formulation.

The distribution and retained amount of benzo[a]pyrene at the micro-zones of mangrove leaf cuticles: Results from a novel analytical method

Plant leaf cuticles play a critical role in the accumulation and transport of atmospheric polycyclic aromatic hydrocarbons (PAHs). The relationship between the distribution and retained amount of PAHs on the leaf cuticles and the leaves micro-zone structures is still unclear. In this study, a confocal microscopic fluorescence spectral analysis (CMFSA) system with a spatial resolution of 200 nm was established as a direct and noninvasive means to determine the microscopic distribution and quantify the retained amount of benzo[a]pyrene (B[a]P) at Aegiceras corniculatum (Ac), Kandelia obovata (Ko) and Avicennia marina (Am) leaf cuticle micro-zones (0.096 mm2). The linear ranges for the established method were 10–1900 ng spot−1 for Ac, 15–1700 ng spot−1 for Ko and 30–1800 ng spot−1 for Am, and the detection limits were 0.06 ng spot−1 for Ac, 0.06 ng spot−1 for Ko and 0.07 ng spot−1 for Am. Notably, B[a]P formed clusters and unevenly distributed at the leaf cuticles. On the adaxial cuticles, B[a]P was mainly accumulated unevenly along the epidermis cell wall, and it was also distinctively distributed in the secretory cells around salt glands for Ac and Am. On the abaxial leaf cuticles, B[a]P was concentrated in the salt glands and stomata apart from being unevenly distributed in the epidermis cell wall. Moreover, the amount of B[a]P retained presented a negative correlation with the polarity of leaf cuticles, which resulted in the amount of B[a]P retained on the adaxial leaf cuticles being significantly higher than that on abaxial leaf cuticles. Our results provide a potential in situ method for investigating the distribution and retained amount of PAHs at plant leaf surface micro-zones, which would contribute to further studying and understanding the mechanism of migration and transformation of PAHs by plant leaves from a microscopic perspective.

Box-Behnken supported development and validation of robust HPTLC method: an application in estimation of punarnavine in leaf, stem, and their callus of Boerhavia diffusa Linn.

Boerhavia diffusa (BD) Linn. (Nyctaginaceae) is one of the most commonly used herbs in the Indian traditional system of medicine for the urinary disorders. The aim of the current investigation was to carry out initiation, development, and maintenance of BD callus cultures and quantitative estimation of punarnavine in plant and callus extracts. Leaves and stem of BD were used as explant for the tissue culture studies using Murashige and Skoog (MS) basal medium. MS Media comprising 2,4-Dichlorophenoxy acetic acid (2,4-D) (1ppm) and 2,4-D (1ppm) + Indole-3-acetic acid (IAA) (1.0ppm) were found to yield friable callus from leaf explant; similarly, 2,4-D (0.3ppm) + IAA (0.75ppm) + Kinetin (0.3ppm) and 2,4-D (0.5ppm) + Naphthalene acetic acid (NAA) (1.5ppm) + Kinetin (0.3ppm) were found to yield friable callus from the stem explant. High-performance thin-layer chromatography method was been developed for the quantitative estimation of punarnavine (R f = 0.73) using mobile phase containing toluene: ethyl acetate: formic acid in the ratio (7.0:2.5:0.7, v/v/v) at 262nm. The validated method was found linear (r 2 = 0.9971) in a wide range (100-1000ng spot-1), precise, accurate, and robust. The values of limit of detection, LOD = 30.3ng spot-1, and limit of quantification, LOQ = 100.0ng spot-1. The robustness of the method was proved by applying the Box-Behnken design (BBD). The developed method found appropriate for the quality control of medicinal plants containing punarnavine as a constituent.

HPTLC method development and validation of stigmasterol from different extracts of Tagetes erecta and Capsicum annuum

A new, simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method for analysis of stigmasterol was developed and validated for the determination of stigmasterol in different extracts. A new, simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method for analysis of stigmasterol was developed and validated for the determination of stigmasterol in different extracts. Analysis of stigmasterol was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of Hexane : Acetone (8:2 v/v) at room temperature. (25 °C ± 2 °C) Camag TLC scanner III was used for spectrodensitometric scanning and analysis in absorbance mode at 490nm. The system was found to give compact spots for stigmasterol. (Rf value of 0.44 ± 0.02) The linear regression analysis data for the calibration plots showed good linear relationship with r2 =0.9997 ± 0.0002 in the concentration range 200-1200 ng spot−1 with respect to peak area. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery and robustness. Statistical analysis of the data showed that the method is reproducible and selective for the estimation of stigmasterol.
 Keywords: Tagetes erecta, Capsicum annuum, stigmasterol, HPTLC, method validation

Stability-indicating high-performance thin-layer chromatographic determination of ondansetron in pure form and pharmaceutical formulations

A new, simple, precise, accurate, stability-indicating, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the determination of ondansetron hydrochloride in pure form and pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of chloroform–methanol–ethyl acetate (7:2:1, v/v). This system was found to give a compact spot of ondansetron (RF value of 0.67 ± 0.011); ondansetron was subjected to acid and alkali hydrolysis, oxidation, and photodegradation. The wavelength of TLC scanner was set at 302 nm for both detection and quantitation. The calibration curves were linear over the range of 25–500 ng spot−1. The limit of detection was 4.9 ng spot−1, and the limit of quantitation was 14.7 ng spot−1. The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines, and the results were acceptable. The propo...

Comparative thin-layer chromatographic studies and development of a high-performance thin-layer chromatography method for the quantification of lawsone in natural and micropropagated plant parts of Lawsonia inermis L.

A simple, sensitive, and precised high-performance thin-layer chromatography (HPTLC) method has been developed for the analysis of lawsone in natural vis-a-vis micropropagated plant parts of Lawsonia inermis L. Separation of the components was perfectly achieved on high-performance thin-layer chromatography (TLC) plates using optimized tertiary mobile phase of benzene‒ethyl acetate‒ acetic acid (7.5:2.5:0.1, v/v). Densitometric scanning was performed before derivatization of the plate in absorption/reflection mode, and lawsone was quantified at its maximum absorbance of wavelength of 275 nm. Linearity of the method was obtained in the concentration range of 50 to 350 ng spot−1 with a correlation coefficient (r2) of 0.9999, indicating good relationship between concentrations in opposition to the peak area. The limit of detection and limit of quantification were found to be 16 and 50 ng spot−1, respectively. The obtained recovery ranges from 95.09% to 96.90% with an average value of 96.02% proved the excell...

Simultaneous quantification of two phenolic biomarkers by a validated high-performance thin-layer chromatographic method in antimicrobial and antioxidant active ethyl acetate fraction of Allium cepa L. (peel)

In this study, we have developed a validated high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of two phenolic biomarkers, protocatechuic acid (compound 1) and quercetin 4ʹ-O-β-d-glucopyranoside (compound 2) in antimicrobial and antioxidant active A. cepa ethyl acetate extract (ACEAE). The quantitative analysis was carried out on normal-phase HPTLC (glass-backed silica gel 60 F254) plates with solvents toluene, ethyl acetate, and formic acid in the ratio of 3:6:1, v/v (as the mobile phase). Well-resolved, compact, and intense peaks of compound 1 (RF = 0.56 ± 0.001) and compound 2 (RF = 0.05 ± 0.001) were found at λmax = 275 nm. The linear regression equation / r2 for compound 1 (Y = 8.89X + 250.71 / 0.994) and compound 2 (Y = 6.64X + 209.34 / 0.998) in the concentration range of 100–700 ng spot−1 indicated good linear relationship. The low values of percent relative standard deviation (%RSD) for intra-day / inter-day precision of compound 1 (1.14–1.26 / 1.08–1....

Development and validation of an HPTLC method for estimation of coronarin D in Hedychium coronarium rhizome

Rhizome extracts of Hedychium coronarium are widely used as phytotherapeutics. As of date, there is no documented study on the standardization of H. coronarium extract, and the following research is an effort in this direction. Coronarin D is an important bioactive compound present in H. coronarium which shows chemopreventive activity against cancer. H. coronarium extracts were assessed for coronarin D content for the first time. The extraction was checked using different solvents: n-hexane, acetone, and methanol. Coronarin D was separated on silica gel 60F254 high-performance thin-layer chromatography (HPTLC) plates by isocratic gradient method using n-hexane-ethyl acetate (80:20 v/v) as mobile phase. Densitometric quantification was performed at 231 nm in absorption mode. This method gave a well-defined peak at Rf 0.20 corresponding to coronarin D. The method was validated using International Conference on Harmonization (ICH) guidelines in terms of precision, repeatability, and accuracy. Linearity range of coronarin D was 200–1000 ng spot−1 with a correlation coefficient of R2 ± SD = 0.9987 ± 2.62% in the concentration range of 200–1000 ng spot−1. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 35 and 115 ng, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels, and the average percentage recovery was found to be 98.22 % for coronarin D. Among the different solvents, acetone produced maximum extraction efficiency of coronarin D. The proposed HPTLC method can be applied for robust identification and quantitative determination of coronarin D in H. coronarium extracts.

Isolation and high-performance thin-layer chromatographic analysis of bioactive lupenone: A lupane triterpenoid from Diospyros melanoxylon

Lupenone was isolated for the first time from the stem bark of Diospyros melanoxylon and characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of lupenone in D. melanoxylon stem bark. HPTLC analysis was performed on HPTLC plates by using a binary mobile phase of n-hexane—ethyl acetate (8.2:1.8, v/v). It was quantified at 395 nm after derivatization with methanol—sulfuric acid reagent. The limits of detection and quantification were found to be 40 ng and 100 ng per spot, respectively. The linear regression analysis data for the calibration plot in the range of 100–500 ng spot−1 showed a good linear relationship between peak area and concentration (r 2 = 0.9997). The instrumental precision was 1.01% (coefficient of variation [CV]) and the repeatability of the method was 2.17% (CV). The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise and has been successfully used for the estimation of lupenone in D. melanoxylon stem bark.

High-performance thin-layer chromatography method for the simultaneous determination of itopride, pantoprazole, and mosapride in their formulations and spiked human plasma

The combination of itopride (ITP), pantoprazole (PAN), and mosapride (MS) is widely used in the treatment of many gastrointestinal tract (GIT) disorders. For that purpose, a new, simple, precise, accurate, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of ITP, PAN, and MS in their pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of methylene chloride–ethyl acetate–methanol– ammonia (25%) (12:2:0.8:0.2, v/v); this system was found to give compact spot of itopride (RF value of 0.22 ± 0.008), pantoprazole (RF value of 0.41 ± 0.006), and mosapride (RF value of 0.62 ± 0.029). The wavelength of thin-layer chromatography (TLC) scanner was set at 289 nm for both detection and quantitation. The calibration curves were linear over the range of 100–1500 ng spot−1 for ITP and MS, and 70–1500 ng spot−1 for PAN. The detection li...

Concurrent analysis of the biologically active markers β-amyrin and β-sitosterol by applying a validated high-performance thin-layer chromatography method in the aerial parts of Tinospora cordifolia and Calotropis gigantia

In this study, we have developed a validated high-performance thin-layer chromatography (HPTLC) method for the concurrent estimation of the biomarkers β-amyrin and β-sitosterol in dichloromethane and ethanol extracts of the aerial parts of Tinospora cordifolia (TCDC and TCET) and Calotropis gigantia (CGDC and CGET). Chromatographic estimations were carried out on HPTLC (glass-backed silica gel 60 F254) plates with solvents hexane and ethyl acetate in the ratio of 7.5:2.5, v/v (as the mobile phase). Post development, the plate was derivatized with p-anisaldehyde reagent to furnish compact spots of β-amyrin and β-sitosterol and scanned at λmax = 530 nm. Well-resolved, compact as well as intense peaks of β-sitosterol (RF = 0.26 ± 0.001) and β-amyrin (RF = 0.39 ± 0.001) were found. The linear regression equation and the correlation coefficient square (r2) for β-amyrin (Y = 6.118x + 460.76 and 0.9959) and β-sitosterol (Y = 7.109x + 1069.1 and 0.9967) in the concentration range of 100–1400 ng spot−1 indicated g...

Chemical fingerprinting and quantification of biomarker wedelolactone in different sources of “Bhringaraja” by high-performance thin-layer chromatography and method validation

Two different botanical sources, Eclipta alba and Wedelia calendulacea are used as “Bhringaraja” in the Ayurvedic s ystem o f medicine. In the present study, an effort has been made to evaluate different sources by using high-performance thin-layer chromatography (HPTLC) as an analytical tool. Wedelolactone, one of the primary constituents of these plants, was taken as the marker compound for the evaluation. An HPTLC method was developed and validated for the evaluation of different sources of “Bhringaraja”. The chromatographic system was developed using silica gel 60 F254 HPTLC plates with the mobile phase toluene–chloroform–ethyl alcohol–formic acid (5:4:1:0.5, v/v). Linearity was found between the concentration ranges of 80 to 280 ng spot−1 (R2: 0.9994), and limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.36 ng spot−1 and 1.09 ng spot−1. The study has shown the presence of wedelolactone at the concentration of 0.26% w/w and 0.05% w/w in E. alba and W. calendulacea, respect...

Intra-specific chemotypic variability of forskolin content inColeus forskohlii(Wild.) Briq. growing in Nilgiri hills of India

Plant metabolite varies with season and geographic conditions. The present study is aimed at the identification of the potential chemotypes of Coleus forskohlii, available in the natural habitat of Nilgiri hills and adjoining area, in order to provide a basic lead for the industry concerning commercial exploitability, including the location-specific commercial cultivation of the plant. The effect of intra-specific variability in the forskolin content among the populations was estimated using high-performance thin-layer chromatography (HPTLC)—densitometric method. The roots of fourteen naturally occurring populations from the entire hill range were collected, covering the wide topography from foot hills up to the highest peak. The method developed for the quantification of forskolin was validated and found to be linear, specific, and accurate with precision and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 1.04 and 3.16 ng spot−1. Precision studies (both inter-day and intra-...

Stability Indicating HPTLC Determination of Triamcinalone Acetonide in Bulk Drug and Sterile Injectable Suspension

A sensitive, accurate, precise and stability indicating high-performance thin layer chromatographic method was developed and validated for analysis of triamcinalone acetonide (TRIA) in bulk drug and sterile injectable suspension. The method employed HPTLC aluminum precoated plates with silica gel 60F-254 as the stationary phase. The solvent system consisted of Toluene: ethyl acetate: ammonia solution (33:67:0.1 %v/v). This system was found to give compact bands for TRIA (R f value 0.38 ± 0.02). TRIA was subjected to acid, alkali and neutral hydrolysis, oxidation, sun light and dry heat treatment. The degraded products were well separated from the pure drug with notably different R f values. CAMAG semi-automatic HPTLC used for the analysis. Densitometric analysis of TRIA was carried out in the absorbance mode at 240 nm. The linear regression data for the calibration plots showed good linear relationship with correlation coefficient 0.9996 ±/ 0.001 in the concentration range of 100.0-2000.0 ng spot -1 . The values of slope and intercept were 9.7841 and -86.13 respectively. The method was validated for precision, accuracy, robustness, and recovery. The limits of detection and quantitation were 33.0 and 100.0 ng spot -1 respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a routine stability indicating assay method. Key words: Triamcinalone Acetonide (TRIA), HPTLC, Stability indicating assay method, Degradation, ICH, Validation.

Concurrent NP-HPTLC Determination of Shikonin and β,β-Dimethylacryl Shikonin in Arnebia benthamii.

Naphthoquinones are important class of molecules found as a natural red color pigments in roots of Arnebia benthamii (Wall. ex G. Don) L M. Johnston. The aim of present investigation is to develop and validates a simple, cost-effective and reliable method for quantification of these compounds. Therefore, a normal phase-high performance thin-layer chromatography (NP-HPTLC) method for concurrent determination of shikonin and β,β-dimethylacryl shikonin in A. benthamii was established. Method development of naphthoquinones in the methanol extract was done using hexane-ethyl acetate-methanol (40:7.5:2.5, v/v/v) solvent system at 520 nm. The developed method showed good band separation for shikonin (Rf, 0.37) and β,β-dimethylacryl shikonin (Rf, 0.58). The linearity ranged between 100 and 8,000 ng spot(-1) with an average recovery of >97% in both cases. The results showed reproducible intraday and interday precision (<2.0% RSD) in quantification of naphthoquinones. The limits of detection are 12.96 and 14.65 ng spot(-1) while the limits of quantification are 39.27 and 44.39 ng spot(-1) for shikonin and β,β-dimethylacryl shikonin, respectively. The developed method is reliable, fast, easy to follow and economic in concurrent assessment of shikonin and β,β-dimethylacryl shikonin in A. benthamii root samples. In addition, it seems to be first report for identification and quantification of β,β-dimethylacryl shikonin from the A. benthamii.

Development of a new, rapid, and sensitive validated high-performance thin-layer chromatographic method for the estimation of berberine inTinospora cordifolia

A straightforward, delicate, and fast elite thin-layer chromatographic system (high-performance thin-layer chromatography [HPTLC]) has been produced and validated for the quantitative determination of berberine in Tinospora cordifolia. The chromatographic development was completed on HPTLC plates precoated with silica gel 60 F254 utilizing a blend of methanol, acetic acid, and water (8:1:1, v/v) as the mobile phase. Detection was completed densitometrically at 366 nm. The RF estimation of berberine was observed to be 0.71 ± 0.02. The system was validated according to the International Conference on Harmonization (ICH) guidelines regarding linearity, precision, accuracy, robustness, and so forth. The calibration curve was observed to be straight over a scope of 120–360 ng spot−1 with a regression coefficient of 0.971. The accuracy was observed to be as high as 98.56%, and the relative standard deviation (% RSD) values for intra-day and between-day variations were under 2%. The system showed high affectabil...

Development of Validated HPTLC Method for Simultaneous Estimation of Fexofenadine Hydrochloride and Montelukast Sodium in Tablet Dosage form

A simple, precise, specific and accurate high performance thin layer chromatographic method has been developed for the simultaneous estimation of Fexofenadine hydrochloride (FEXO) and Montelukast sodium (MONT) in pharmaceutical dosage form. The separation was carried out on Merck HPTLC aluminium plates of silica gel G60 F254, (20 × 10 cm) with 250 μm thickness using hexane: ethyl acetate: propanol (2: 5: 3, v/v/v) as mobile phase. Densitometric measurement was carried out in the absorbance mode at 230 nm. The drugs were resolved satisfactorily with R values of 0.31 ± 0.01 and 0.57 ± 0.01 for FEXO and MONT, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r2=0.9996 and 0.9998 for FEXO and MONT, respectively in the concentration range of 1200–6000 ng spot−1 for FEXO and 100–500 ng spot−1 for MONT. The method was validated for precision, robustness, specificity and accuracy. The limit of detection and quantitation were 393 and 1193 ng spot−1, respectively for FEXO and 37 and 112 ng spot−1, respectively for MONT. The proposed method can be successfully used to determine the drug content of marketed formulation.

Chromatographic Determination of Aminoacridine Hydrochloride, Lidocaine Hydrochloride and Lidocaine Toxic Impurity in Oral Gel

Two sensitive and selective analytical methods were developed for simultaneous determination of aminoacridine hydrochloride and lidocaine hydrochloride in bulk powder and pharmaceutical formulation. Method A was based on HPLC separation of the cited drugs with determination of the toxic lidocaine-related impurity 2,6-dimethylaniline. The separation was achieved using reversed-phase column C18, 250 × 4.6 mm, 5 µm particle size and mobile phase consisting of 0.05 M disodium hydrogen phosphate dihydrate (pH 6.0 ± 0.2 adjusted with phosphoric acid) and acetonitrile (55 : 45, v/v). Quantitation was achieved with UV detection at 240 nm. Linear calibration curve was in the range of 1.00-10.00, 13.20-132.00 and 1.32-13.20 µg mL(-1) for aminoacridine hydrochloride, lidocaine hydrochloride and 2,6-dimethylaniline, respectively. Method B was based on TLC separation of the cited drugs followed by densitometric measurement at 365 nm on the fluorescent mode for aminoacridine hydrochloride and 220 nm on the absorption mode for lidocaine hydrochloride. The separation was carried out using ethyl acetate-methanol-acetic acid (65 : 30 : 5 by volume) as a developing system. The calibration curve was in the range of 25.00-250.00 ng spot(-1) and 0.99-9.90 µg spot(-1) for aminoacridine hydrochloride and lidocaine hydrochloride, respectively. The results obtained were statistically analyzed and compared with those obtained by applying the manufacturer's method.

A validated high-performance thin-layer chromatographic method for the quantitation of protodioscin inTrigonella foenum-graecumand its herbal formulations

A simple, rapid, and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of protodioscin from Trigonella foenum-graecum. Chromatographic separation was achieved on aluminum plates precoated with silica gel G60 F254 as the stationary phase and chloroform–methanol–glacial acetic acid (6.5:3.0:0.5 v/v) as the mobile phase. Densitometric evaluation was carried out at 515 nm after derivatizing the spots with p-dimethyl aminobenzaldehyde. The RF value of the protodioscin was found to be 0.31 ± 0.02. The response in terms of peak area was found to be linear over the concentration range from 400 to 1400 ng spot−1 with a regression coefficient of 0.9981 and mean recovery of 100.90 ± 3.00%. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. The limit of detection and limit of quantitation were found to be 100 ng spot−1 and 400 ng spot−1, respectively. Robustness of the developed method was assessed using experim...

TLC—Densitometric Determination of Sulfasalazine and Its Possible Impurities in Pharmaceutical Preparations

A simple, selective, and sensitive thin-layer chromatographic—densitometric method has been developed for the determination of sulfasalazine besides its possible impurities in pharmaceutical preparations. The mobile phase was composed of ethyl acetate—methanol—ammonia 25% 10:7:3 (υ/υ/υ), and the stationary phase was aluminum plates precoated with silica gel 60 F254 that enabled to obtain well resolved peaks of sulfasalazine and its impurities. The developed chromatograms were analyzed densitometrically at λ = 360 nm. RF values and ultraviolet (UV) spectra were used to identify the compounds. The developed method is highly sensitive (limit of detection [LOD] = 17.11 ng spot−1, limit of quantitation [LOQ] = 51.84 ng spot−1), precise (relative standard deviation [RSD] = 1.43%–4.28%), and accurate (RSD = 1.64%–4.27%). The linearity of the method was checked within the range 20–120 ng spot−1. The method was successfully applied for the determination of sulfasalazine in pharmaceutical preparations besides its i...